Figure 1

Identification of compounds with autophagy-regulating activity and repurposing potential for HDT against Mtb and Stm. (A) MelJuSo-Mtb model-based screening results of the Screen-Well Autophagy library at 10 µM concentration after 24 h of treatment sorted by effect size on the percentage of infected cells (DsRed-Mtb positive cells) compared to DMSO. Data points display the mean ± standard deviation of 3 replicates. The positive control H-89 and two structural analogues Fluspirilene and Pimozide are annotated. (B) MelJuSo-Mtb model-based screening results of H-89, Fluspirilene and Pimozide at 10 µM concentration after 24 h of treatment on the percentage of cells compared to DMSO. Data points display the mean of 3 replicates and represent two independent experiments. Dotted lines indicate DMSO set at 100% and median is shown for each condition. Statistical significance was tested using a RM one-way ANOVA with Dunnett’s multiple comparison test (* = p-value < 0.05). (C) CFU assay of Mφ1 (upper panel) and Mφ2 (lower panel) infected with Mtb and treated with 10 µM of Fluspirilene, Pimozide, H-89 as positive control or DMSO at equal v/v for 24 h. Effect of compound treatments are shown separate since donors tested were not always identical. Each dot represents a single donor (7 and 8 for H-89, 8 and 9 for Fluspirilene and 9 and 10 donors for Pimozide were tested in Mφ1 and Mφ2, respectively) and depicts the mean of 3 or 4 replicates. Dotted lines indicate DMSO set at 100% and median + 95% confidence intervals are shown for every condition. Statistical significance was tested using Wilcoxon matched-pairs signed rank test with post-hoc Benjamini–Hochberg correction (right panel) (* = q-value < 0.1). (D) CFU assay of Mφ1 (upper panel) and Mφ2 (lower panel) infected with Stm and treated with 10 µM of Fluspirilene, Pimozide, H-89 as positive control or DMSO at equal v/v for 24 h. Each dot represents a single donor (6 donors in total) and depicts the mean of 3 or 4 replicates. Dotted lines indicate DMSO set at 100% and median + 95% confidence intervals are shown for every condition. Statistical significance was tested using Wilcoxon matched-pairs signed rank test with post-hoc Benjamini–Hochberg correction (right panel) (* = q-value < 0.1). (E) Mtb growth in liquid culture during treatment with 10 μM of Fluspirilene, Pimozide or DMSO at equal v/v at assay endpoint, day 10. Rifampicin (20 μg/ml) was used as positive control for Mtb growth inhibition. Bars depict mean ± standard deviation of 3 replicates. Experiment shown is a representative of 4 independent experiments. Statistical significance of treatment versus DMSO was tested using a one-way ANOVA with Dunnett’s multiple comparisons test (**** = p-value < 0.0001). (F) Stm growth in liquid culture during treatment with 10 μM of Fluspirilene, Pimozide or DMSO at equal v/v. Gentamicin (50 μg/ml) was used as positive control for Stm growth inhibition. Bars depict mean ± standard deviation of 3 replicates. Experiment shown is a representative of 2 independent experiments. Statistical significance of treatment versus DMSO was tested using a one-way ANOVA with Dunnett’s multiple comparisons test (**** = p-value < 0.0001).